OBJECTIVETo investigate the main proteinases responsible for CD16b shedding under different stimulators.

METHODSHEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation.

RESULTSHEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA.

CONCLUSIONSBoth ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.

ADAM Proteins ; genetics ; metabolism ; physiology ; ADAM10 Protein ; ADAM17 Protein ; Amyloid Precursor Protein Secretases ; genetics ; metabolism ; physiology ; Calcium Ionophores ; pharmacology ; Carcinogens ; pharmacology ; Cells, Cultured ; Drug Evaluation, Preclinical ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ionomycin ; pharmacology ; Membrane Proteins ; genetics ; metabolism ; physiology ; Protein Processing, Post-Translational ; drug effects ; Protein Transport ; drug effects ; Proteolysis ; drug effects ; Receptors, IgG ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology ; Transfection

OBJECTIVETo investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.

METHODSPhorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.

RESULTSPMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.

CONCLUSIONSSphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.

Apoptosis ; drug effects ; Carcinogens ; administration & dosage ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; administration & dosage ; pharmacology ; HT29 Cells ; Humans ; MAP Kinase Kinase 4 ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; physiology ; Sphingosine ; administration & dosage ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; administration & dosage ; analogs & derivatives ; pharmacology ; Time Factors ; p38 Mitogen-Activated Protein Kinases ; metabolism

The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type-Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer.
Proto-Oncogene Proteins c-met/*metabolism/*physiology ; Protein Isoforms/metabolism/physiology ; NIH 3T3 Cells ; Mutant Proteins/metabolism/physiology ; Mice, Nude ; Mice ; Hepatocyte Growth Factor/pharmacology ; Female ; Down-Regulation ; Carcinogens/*metabolism ; Carcinogenicity Tests ; Animals ; *Alternative Splicing

An environmental pollutant, tetrachloro dibenzo dioxin (TCDD) is known to illicit the cognitive disability and motor dysfunction in the developing brain. TCDD induced effects leading to neurodevelopmental and neurobehavioral deficit may have been defined, however underlying molecular mechanism and possible intracellular targets remain to be elucidated. In this study, we attempted to analyze TCDD-induced neurotoxic effects in the granule cells from cerebellum where certain cognitive abilities and motor function command are known to be excuted. [3H]PDBu, (phorbol 12,13-dibutyrate) binding assay indicated that TCDD induced a dose-dependent increase of total PKC activity and its induction was the aryl hydrocarbon receptor (AhR) dependent and N-methyl-D-aspartate receptor (NMDAR) independent. TCDD also caused the translocation of both PKC-alpha and -epsilon in a dose-dependent manner but associated with different receptors; PKC-alpha via AhR but not PKC-epsilon indicating an isozyme-specific pattern of the induction. Increase of the ROS formation was also observed in the cells treated with TCDD in a dose-dependent and an AhR-dependent manner. The treatment of the cells with the diamino dicyano-bis(2-aminophenylthio) butadiene (U0126, MEK-1/2 inhibitor), dizocilpine maleate (MK-801, non-competitive N-methyl-D-aspartate glutamate receptor antagonist) and vitamin E attenuated the TCDD-induced ROS production indicating that TCDD-induced ROS formation may be associated with activation of ERK-1/2 in the MAP kinase pathway or the NMDA receptor. TCDD also increased [Ca2+]i, which is associated with ROS formation and PKC activation in the cerebellar granule cells. It is suggested that TCDD activates the NMDA receptor, which may induce a sustained increase of [Ca2+]i in neurons followed by the ROS formation. Our findings may contribute to understanding the mechanism of TCDD-related neurotoxicity, thereby improving the health risk assessment of neurotoxic compounds in humans.
Animals ; Binding, Competitive ; Butadienes/pharmacology ; Carcinogens/pharmacology ; Cerebellum/cytology/*drug effects/enzymology ; Dizocilpine Maleate/pharmacology ; Environmental Pollutants/*toxicity ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism ; Neuroprotective Agents/pharmacology ; Nitriles/pharmacology ; Phorbol 12,13-Dibutyrate/pharmacology ; Protein Kinase C/*metabolism ; Protein Transport ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Receptors, Aryl Hydrocarbon/metabolism ; Receptors, N-Methyl-D-Aspartate/metabolism ; Research Support, Non-U.S. Gov't ; Tetrachlorodibenzodioxin/*toxicity

OBJECTIVETo screen the over differentially expressed genes in carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C cells).

METHODSThe suppression subtractive hybridization (SSH) method was performed to profile differentially expressed genes between 16HBE-C cells and 16HBE cells. The cDNA fragments of differentially expressed genes were inserted into TA cloning vector and transformed competent E. coli strain. Positive clones were randomly picked up and identified by the colony PCR method. Dot blot was used to test the same source with the tester. The differentially expressed cDNA fragments were sequenced and compared with known genes and EST database in Genbank.

RESULTSEight known genes were over-expressed in 16HBE-C cells including eukaryotic translation elongation factor 1 alpha 1, HIF-1 responsive RTP801, ribosomal protein L10 (RPL10), ribosomal protein S29 (RPS29), mitochondrion related genes, and laminin receptor 1. Three differentially expressed cDNA fragments could not be matched to the known genes but to the EST database.

CONCLUSIONThe SSH method can detect differentially expressed genes between 16HBE-C and 16HBE cells. BPDE-induced carcinogenesis may be related to alteration of at least eight known genes and three unknown genes. These expression data provide a clue to further cloning novel genes and studying functions in BPDE-induced carcinoma.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide ; pharmacology ; toxicity ; Carcinogens ; pharmacology ; toxicity ; Carcinoma ; genetics ; metabolism ; Cell Line ; Cell Line, Transformed ; Cell Transformation, Neoplastic ; chemically induced ; Gene Expression Regulation, Neoplastic ; Humans ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; RNA, Messenger ; metabolism

Evidences show that eukaryotic mRNAs can perform protein translation through internal ribosome entry sites (IRES). 5'-Untranslated region of the mRNA encoding apoptotic protease-activating factor 1 (Apaf-1) contains IRES, and, thus, can be translated in a cap-independent manner. Effects of changes in protein translation pattern through rapamycin pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-butanone(NNK, tobacco-specific lung carcinogen)-induced apoptosis in human bronchial epithelial cells were examined by caspase assay, FACS analysis, Western blotting, and transient transfection. Results showed that NNK induced apoptosis in concentration- and time-dependent manners. NNK-induced apoptosis occurred initially through cap-independent protein translation, which during later stage was replaced by cap-dependent protein translation. Our data may be pplicable as the mechanical basis of lung cancer treatment.
Antibiotics, Antineoplastic/pharmacology ; Apoptosis/*drug effects ; Apoptotic Protease-Activating Factor 1 ; BH3 Interacting Domain Death Agonist Protein ; Blotting, Western ; Bronchi/metabolism/*pathology ; Carcinogens/*pharmacology ; Carrier Proteins/metabolism ; Caspases/metabolism ; Cytochromes c/metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells/metabolism/*pathology ; Eukaryotic Initiation Factor-4E/metabolism ; Flow Cytometry ; Humans ; Nitrosamines/*pharmacology ; Protein Biosynthesis ; Proteins/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA Cap-Binding Proteins/*physiology ; Sirolimus/pharmacology ; Time Factors ; bcl-2-Associated X Protein

OBJECTIVE12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.

METHODSIncubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.

RESULTSAfter treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).

CONCLUSIONK562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinogens ; pharmacology ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Division ; drug effects ; genetics ; Cell Membrane ; chemistry ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factors ; genetics

BACKGROUNDTo study the effect of human papillomavirus (HPV) 16 E6/E7 and TPA (12-O-tetradecanog-1-phorbol-13-acetate) on malignant transformation of human embryo oral tissue.

METHODSRecombinant plasmid with HPV 16 E6/E7 was constructed and transfected into human embryo oral tissue. The oral tissue with HPV 16 E6/E7 gene or without the gene was inoculated into the hypophloeodal of right shoulder in scid mice, respectively. The study was conducted in four groups: the first group was the oral tissue transfected plasmid with HPV 16 E6/E7 plus TPA, which were inoculated into 8 scid mice; the second group was only oral tissue transfected with plasmid with HPV 16 E6/E7 into 6 scid mice; the third group was normal oral tissue plus TPA inoculated into 6 scid mice, and the final group was only normal oral tissue inoculated into 5 scid mice. Three days after inoculation, TPA was injected at the left shoulder of the mice once a week. Twelve weeks after inoculation, tumor was found in 7 scid mice from the first group. HPV 16 E6/E7 gene in tumor tissues was analyzed by PCR.

RESULTSThe rate of tumor formation was 7/8 in the first group; no tumor was found in the other groups. Pathological diagnosis of the tumor was fibrohistiocytoma. HPV 16 E6/E7 gene was detected by PCR in tumor tissues.

CONCLUSIONWith the cooperating action of TPA, human oral tissue containing HPV 16 E6/E7 gene could cause malignant transformation in scid mice.

Animals ; Carcinogens ; pharmacology ; Carcinoma ; pathology ; virology ; Cell Transformation, Neoplastic ; drug effects ; Cells, Cultured ; Human papillomavirus 16 ; genetics ; metabolism ; Humans ; Mice ; Mice, SCID ; Mouth Neoplasms ; pathology ; virology ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus E7 Proteins ; genetics ; metabolism ; Papillomavirus Infections ; pathology ; virology ; Repressor Proteins ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo establish an in vitro heterogeneous expression model of human CYP2E1 (hCYP2E1) cDNA and investigate the effect of the chemical carcinogenic N, N'-dinitrosopiperazine (DNP) on the expression of CYP2E1.

METHODSExogenous hCYP2E1 was introduced into the mouse derived NIH3T3 cells using the lipofectamine transfection technique. Integration of exogenous hCYP2E1 gene was identified by PCR and Southern blot. After treatment with various concentration of ethanol and DNP on the transfected NIH3T3 cell cultures, RT-PCR and Western blot was applied to detect the expression level of CYP2E1.

RESULTSTwo cell clones with integration and stable expression of exogenous hCYP2E1 were obtained and designated as NIH3T3-2E1-A4 and NIH3T3-2E1-A8 respectively. The expression of both hCYP2E1 mRNA and protein products was promoted after either ethanol or DNP treatment.

CONCLUSIONThe results suggested that the promoted expression of hCYP2E1 induced by DNP and /or ethanol is due to enhanced transcription. The mechanism of DNP carcinogenes is might be related to this in situ activated metabolism by CYP2E1.

3T3 Cells ; Animals ; Carcinogens ; toxicity ; Cytochrome P-450 CYP2E1 ; genetics ; Ethanol ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Mice ; Nitrosamines ; toxicity ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo examine the possible role of aristolochic acid (AA) in transdifferentiation and apoptisis of human tubular epithelial cell line (HKC).

METHODSCultured HKC cells were divided into five groups: serum-free (negative control) and treatment with AA at the concentrations of 5 mg/L, 10 mg/L, 20 mg/L and 40 mg/L for 48 hours, respectively. Transdifferentiation of HKC cells was observed with the following methods: detection of the expression of vimentin and cytokeratin of HKC cells with indirect immunoflourescence, determination of expression of E-cadherin and alpha-smooth muscle actin (alpha-SMA) by indirect immunohistochemical double staining, and determination of the proportion of alpha-SMA (+) HKC cells by flow cytometry. The apoptosis of HKC cells was observed with Giemsa staining, TUNEL reaction and agarose gel electrophoresis, and the ratio of apoptotic HKC cells was quantitatively analyzed by flow cytometry with propidium iodide staining.

RESULTSThe expression of cytokeratin and E-cadherin reduced and that of vimentin increased in HKC cells treated with 10 mg/L of AA for 48 hours, and the expression of alpha-SMA (+) in HKC cells treated with 10 mg/L of AA (14.17 +/- 0.61)% was significantly higher than that in serum-free controls (3.57 +/- 0.52)%. Apoptosis of HKC cell treated with 40 mg/L of AA for 48 hours was 53.4%, significantly higher than that in serum-free controls (2%). Treatment with 5 mg/L of AA and 20 mg/L of AA could not induce apoptosis and transdifferentiation of cells.

CONCLUSIONSTreatment with relatively low concentration of AA (10 mg/L) might induce slight transdifferentiation in cultured HKC cells and that with higher concentration of AA (40 mg/L) for 48 hours might induce apparent apoptosis of these cells, which suggested that transdifferentiation and apoptosis of tubular epithelial cells probably played important roles in aristolochic acid-induced nephropathy.

Actins ; analysis ; Apoptosis ; drug effects ; genetics ; Aristolochic Acids ; pharmacology ; Carcinogens ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line ; DNA Fragmentation ; drug effects ; Epithelial Cells ; drug effects ; metabolism ; ultrastructure ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Kidney Tubules ; cytology ; drug effects ; ultrastructure ; Microscopy, Electron ; Muscle, Smooth ; chemistry